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  • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
  • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
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  • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
  • C07D487/04—Ortho-condensed systems

Definitions

• the present invention is directed to compounds, their synthesis, and their use as modulators or inhibitors of the IL-2 inducible T-cell kinase ("ITK”), which belongs to the TEC family of non-receptor tyrosine kinases essential for T cell activation.
• ITK inducible T-cell kinase
• the present invention is also directed to compounds, their synthesis, and their use as modulators or inhibitors of the Janus Kinase 3 (“JAK3”), which is one of the Janus Kinases (“JAKs”): JAK1, JAK2, JAK3 and TYK2.
• JAKs are signal transducers and activators of transcription ("STAT") family of transcription factors that play key roles in cytokine-induced signal transduction leading to IL-2, IL-4, IL-7, IL-9 and IL-15 release.
• STAT transcription
• the present invention is directed to 3,5-(un)substituted-lH-pyrrolo[2,3-/?]pyridine, lH-pyrazolo[3,4- &]pyridine and 5H- pyrrolo[2,3-£]pyrazine compounds that are dual ITK and JAK3 kinase inhibitors.
• ITK plays a central role in signaling through antigen-receptors; TCR and collectively with co-stimulation of CD4 and CD28, the TCR will trigger a cascade of signal transduction events.
• Tec kinase family of kinases includes; ITK, TEC, BMX, BTK,
• ITK is highly expressed in inflammatory T-cells, NK and mast cells and communicate signals to downstream effectors, including PLC- ⁇ .
• TEC family of kinases play prominent role in T-cell proliferation and the release of cytokines such as IL-2, IL-4, IL-5, IL-lO and IL-13 and IFN- ⁇ .
• JAKs Janus Kinases
• JAK1 JAK2, JAK3 and TYK2 are tyrosine kinases associated to common chain signaling to intracellular effector pathways and are signal transducer and activator of transcription (STAT) family of transcription factors.
• STAT signal transducer and activator of transcription
• the cytokine receptor binding stimulates the recruitment of JAKs, which is autophosphorylated. JAKs then phosphorylate the receptor, and a STAT protein binds to the phosphorylated receptors (SRC homology 2 (SH) domain) leading to the phosphorylation of STATs by JAKs.
• SRC homology 2 (SH) domain phosphorylated receptors
• Phosphorylated STAT proteins in turn dimerize and then translocate to the nucleus in order to regulate gene expression.
• Blocking and or targeting of the JAK-STAT pathway have been shown to be efficacious in clinical trials, with the successful use of JAK kinase inhibitors, for treating of patients with rheumatoid arthritis ("RA").
• Non-selective JAK inhibitors or lack of JAK3 selective inhibitors has delayed the role of JAK3 in autoimmune disorders.
• a selective JAK3 inhibitor has the potential benefit of relieving adverse effects of JAK 1 and JAK2 inhibition such as hematopoiesis and dyslipidemia. Thus, there is a great need for such selective JAK3 inhibitors.
• the compounds of the present invention are covalent/irreversible and reversible inhibitors useful for modulating (e.g. inhibiting) ITK and JAK3 activity for treating diseases or conditions mediated by ITK and JAK3 such as, for example, disease states associated with abnormal cell growth such as autoimmune, inflammation, rheumatoid arthritis and cancer diseases.
• the present invention also provides pharmaceutically acceptable compositions comprising the compounds of the invention and methods of using the compositions in the treatment of various autoimmune, inflammatory, metabolic and cancer disorders.
• the invention also provides processes for preparing the compounds of the invention.
• Non-receptor tyrosine kinase ITK and Janus kinases are key regulators of cytokine pathways and are important targets of therapeutic value in both inflammatory/RA and Cancer/Myeloproliferative diseases.
• Selective small-molecule inhibitors of both ITK and JAK3 is a challenging due to the highly conserved ATP binding pocket within the TEC-kinase family; ITK, TEC, BMX, BTK and TXK/RLK and Janus family members; JAK1, JAK2, JAK3 and TYK2.
• FFDDTM Fraction Field Drug Design
• This methodology assisted in fragments, scaffolds to lead compounds, and subsequent screening and SAR efforts; we have discovered the present first-in-class 3,5-(un)substituted- lH-pyrrolo[2,3-/7]pyridine, lH-pyrazolo[3,4-/?]pyridine and 5H- pyrrolo[2,3-Z?]pyrazine dual ITK and JAK3 inhibitors claimed here, useful for treating multiple disease indications, including autoimmune diseases; more specifically rheumatoid arthritis and other disease indications such as inflammatory, hyperproliferative, or immunologically-mediated diseases.
• the present invention encompassing administering to a human patient a compound of the present invention.
• the compounds may be in a composition as a single dosage form or as part of a multiple dosage forms.
• the present invention includes the use of the compounds herein to treat rheumatoid arthritis, psoriasis, lupus erythematosus, systemic lupus erythematosus, artherosclerosis, idiopathic thrombocytopenia purpura, restenosis, angioplasty, tumours, artherosclerosis, systemic lupus erythematosus, chronic allograft rejection and acute allograft rejection (including from transplantation of heart, liver, kidney, lung, bone marrow, skin and cornea), chronic graft versus host diseases, asthma, allergic acute rhinitis, psoriatic arthritis, systemic sclerosis, atopical dermatitis, erythemas, Alopecia, multiple sclerosis, artherosclerosis and plethora of diseases including immunodeficiencies, myeloproliferative disorders and cancer (acute leukemia, gain of function mutations associated with inherited polycythaemia) diseases
• the present invention concerns compounds active on protein kinases, specifically ITK and JAK3, including mutations of these kinases and their use in treating disease and conditions associated with regulations of the activity of these kinases. More specifically the invention concerns compounds of Formula I as described below. Thus the invention provides use of novel compounds for therapeutic methods involving inhibition and/or modulation of protein kinases ITK and JAK3.
• compositions or salts thereof their synthesis and their use as ITK and JAK3 inhibitors including such compounds and methods of their use in the treatment of various diseases and disorders such as autoimmune diseases.
• the present invention relates to compounds according to Formula I and its subgenus Formulas IA, IB and IC below and pharmaceutically acceptable compositions and salts thereof, their synthesis and their use as ITK and JAK3 inhibitors including such compounds and methods of their use in the treatment of various diseases and disorders such as rheumatoid arthritis, psoriasis, inflammation, hyperproliferative diseases, or
• immunologically-mediated diseases and encompasses administering such compounds to a human disease patient.
• L 1 is H, halo, d_ 4 alkyl, -NH-S(0) 2 (Ci_ 4 alkyl ), or phenyl optionally substituted with 1-3 substituents, each substituent independently selected from halo, Ci_ 4 alkyl, Ci_ 4 alkoxy, -CO-0-Ci- 4 alkyl, -CO-N(Ci- 4 alkyl)( C
• R 1 is each independently H, halo, Ci_ 4 alkyl, or Ci_ 4 alkoxy;
• Z is , optionally substituted with 1-3 independent halo or Ci_ 4 alkyl substituents
• R 2 is each independently H, halo, Ci_ 4 alkyl, cyano-Ci_ 4 alkyl,
• n 0, 1, 2 or 3;
• n 0, 1, or 2; provided that the compound is not
• X 1 is N
• X 2 is CH
• L 1 is ,
• compounds of the present invention are described by Formula (I) and pharmaceutically acceptable salts thereof, wherein X 1 is N, X 2 is CH, L 1 is phenyl optionally substituted with 1-3 substituents, each substituent independently selected from halo, C 1-4 alkyl, Ci_ 4 alkoxy, -CO-0-Ci- 4 alkyl, -CO-
• compounds of the present invention are described by Formula (I) and pharmaceutically acceptable salts thereof, wherein X 1 is N, X 2 is CH, L 1 is phenyl optionally substituted with 1-3 substituents, each substituent independently selected from halo, Ci_ 4 alkyl, Ci_ 4 alkoxy, -CO-0-Ci_ 4 alkyl, -CO-N(Ci_ 4 alkyl)(
• Ci_ 4 alkyl O O , o ⁇ O C H 3 ) a nd H 3 C CH3 , Z is phenyl, and the other variables are as defined above for Formula (I).
• Compounds of the present invention include:
• X 1 is CH
• X 2 is CH
• Z is phenyl
• L 1 is , and the other variables are as defined above for Formula (I).
• compounds of the present invention are described by Formula (I) and pharmaceutically acceptable salts thereof, wherein above for Formula (I).
• X is CH, X is CH, Z is L is H, and the other variables are as defined above for Formula (I).
• X 1 is CH
• X 2 is CH
• Z i and the other variables are as defined above for Formula (I).
• Compounds of the present invention include:
• Compounds of the present invention include the compound:
• Alkyl refers to a saturated straight or branched hydrocarbon radical of one to six carbon atoms, preferably one to four carbon atoms, e.g., methyl, ethyl, propyl, 2-propyl, n- butyl, iso-butyl, tert-butyl, pentyl, hexyl, and the like, preferably methyl, ethyl, propyl, or 2- propyl.
• saturated straight chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like. Cyclic alkyls are referred to herein as a
• Unsaturated alkyls contain at least one double or triple bond between adjacent carbon atoms (referred to as an "alkenyl” or “alkynyl”, respectively.)
• alkenyl or “alkynyl”, respectively.
• Representative straight chain and branched alkenyls include ethylenyl, propylenyl, 1-butenyl, 2-butenyl,
• Co- 4 alkyl refers to an alkyl with 0, 1, 2, 3, or 4 carbon atoms. Co- 4 alkyl with 0 carbon atoms is a hydrogen atom when terminal and is a direct bond when linking.
• Alkylene means a linear saturated divalent hydrocarbon radical of one to six carbon atoms or a branched saturated divalent hydrocarbon radical of three to six carbon atoms, e.g., methylene, ethylene, 2,2-dimethylethylene, propylene, 2-methylpropylene, butylene, pentylene, and the like, preferably methylene, ethylene, or propylene.
• Cycloalkyl refers to a saturated cyclic hydrocarbon radical of three to eight carbon atoms, e.g., cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.
• Alkoxy means a radical -OR a where R a is an alkyl as defined above, e.g., methoxy, ethoxy, propoxy, butoxy and the like.
• Halo means fluoro, chloro, bromo, or iodo, preferably fluoro and chloro.
• Haloalkyl means alkyl substituted with one or more, preferably one, two or three, same or different halo atoms, e.g., -CH 2 C1, -CF 3 , -CH 2 CF 3 , -CH 2 CCI 3 , and the like.
• Haloalkoxy means a radical -OR b where R b is an haloalkyl as defined above, e.g., trifluoromethoxy, trichloroethoxy, 2,2-dichloropropoxy, and the like.
• Acyl means a radical -C(0)R c where R c is hydrogen, alkyl, or haloalkyl as defined herein, e.g., formyl, acetyl, trifluoroacetyl, butanoyl, and the like.
• Aryl refers to an all-carbon monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms) groups of 6 to 12 carbon atoms having a completely conjugated pi-electron system. Examples, without limitation, of aryl groups are phenyl, naphthyl and anthracenyl. The aryl group may be substituted or unsubstituted.
• substituted aryl refers to the aryl group being substituted with one or more, more preferably one, two or three, even more preferably one or two substituents independently selected from the group consisting of alkyl (wherein the alkyl may be optionally substituted with one or two substituents), haloalkyl, halo, hydroxy, alkoxy, mercapto, alkylthio, cyano, acyl, nitro, phenoxy, heteroaryl, heteroaryloxy, haloalkyl, haloalkoxy, carboxy, alkoxycarbonyl, amino, alkylamino dialkylamino, aryl, heteroaryl, carbocycle or heterocycle (wherein the aryl, heteroaryl, carbocycle or heterocycle may be optionally substituted).
• Heteroaryl refers to a monocyclic or fused ring (i.e., rings which share an adjacent pair of atoms) group of 5 to 12 ring atoms containing one, two, three or four ring heteroatoms selected from N, O, or S, the remaining ring atoms being C, and, in addition, having a completely conjugated pi-electron system.
• heteroaryl groups examples, without limitation, of unsubstituted heteroaryl groups are pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline, purine, triazole, tetrazole, triazine, and carbazole.
• the heteroaryl group may be unsubstituted or substituted, such as, for example, 5-methylthiazolyl.
• substituted heteroaryl refers to the heteroaryl group being substituted with one or more, more preferably one, two or three, even more preferably one or two substituents independently selected from the group consisting of alkyl (wherein the alkyl may be optionally substituted with one or two substituents), haloalkyl, halo, hydroxy, alkoxy, mercapto, alkylthio, cyano, acyl, nitro, haloalkyl, haloalkoxy, carboxy, alkoxycarbonyl, amino, alkylamino dialkylamino, aryl, heteroaryl, carbocycle or heterocycle (wherein the aryl, heteroaryl, carbocycle or heterocycle may be optionally substituted).
• Carbocycle refers to a saturated, unsaturated or aromatic ring system having 3 to 14 ring carbon atoms.
• the term “carbocycle”, whether saturated or partially unsaturated, also refers to rings that are optionally substituted.
• the term “carbocycle” includes aryl.
• the term “carbocycle” also includes aliphatic rings that are fused to one or more aromatic or nonaromatic rings, such as in a decahydronaphthyl or tetrahydronaphthyl, where the radical or point of attachment is on the aliphatic ring.
• the carbocycle group may be substituted or unsubstituted.
• substituted carbocyle refers to the carbocycle group being substituted with one or more, more preferably one, two or three, even more preferably one or two substituents independently selected from the group consisting of alkyl (wherein the alkyl may be optionally substituted with one or two substituents), haloalkyl, halo, hydroxy, alkoxy, mercapto, alkylthio, cyano, acyl, nitro, haloalkyl, haloalkoxy, carboxy, alkoxycarbonyl, amino, alkylamino dialkylamino, aryl, heteroaryl, carbocycle or heterocycle (wherein the aryl, heteroaryl, carbocycle or heterocycle may be optionally substituted).
• Heterocycle refers to a saturated, unsaturated or aromatic cyclic ring system having 3 to 14 ring atoms in which one, two or three ring atoms are heteroatoms selected from N, O, or S(0) m (where m is an integer from 0 to 2), the remaining ring atoms being C, where one or two C atoms may optionally be replaced by a carbonyl group.
• heterocycle includes heteroaryl.
• substituted heterocyclyl refers to the heterocyclyl ring being substituted independently with one or more, preferably one, two, or three substituents selected from alkyl (wherein the alkyl may be optionally substituted with one or two substituents), haloalkyl, cycloalkylamino,
• cycloalkylalkyl cycloalkylaminoalkyl, cycloalkylalkylaminoalkyl, cyanoalkyl, halo, nitro, cyano, hydroxy, alkoxy, amino, alkylamino, dialkylamino, hydroxyalkyl, carboxyalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, aralkyl, heteroaralkyl, aryl, heteroaryl, carbocycle, heterocycle (wherein the aryl, heteroaryl, carbocycle or heterocycle may be optionally substituted), aralkyl, heteroaralkyl, saturated or unsaturated heterocycloamino, saturated or unsaturated heterocycloaminoalkyl, and -CORa (where 3 ⁇ 4 is alkyl).
• heterocyclyl includes, but is not limited to, tetrahydropyranyl, 2,2- dimethyl-l,3-dioxolane, piperidino, N-methylpiperidin-3-yl, piperazino, N-methylpyrrolidin- 3-yl, pyrrolidino, morpholino, 4-cyclopropylmethylpiperazino, thiomorpholino,
• thiomorpholino- 1 -oxide thiomorpholino- 1 , 1 -dioxide, 4-ethyloxycarbonylpiperazino, 3- oxopiperazino, 2-imidazolidone, 2-pyrrolidinone, 2-oxohomopiperazino,
• the heterocycle group is optionally substituted with one or two substituents independently selected from halo, alkyl, alkyl substituted with carboxy, ester, hydroxy, alkylamino, saturated or unsaturated heterocycloamino, saturated or unsaturated heterocycloaminoalkyl, or dialkylamino.
• heterocyclic group optionally substituted with an alkyl group means that the alkyl may but need not be present, and the description includes situations where the heterocycle group is substituted with an alkyl group and situations where the heterocycle group is not substituted with the alkyl group.
• stereoisomers Stereoisomers that are not mirror images of one another are termed
• enantiomers and those that are non-superimposable mirror images of each other are termed "enantiomers".
• enantiomers When a compound has an asymmetric center, for example, it is bonded to four different groups; a pair of enantiomers is possible.
• An enantiomer can be
• the compounds of this invention may possess one or more asymmetric centers; such compounds can therefore be produced as individual (R)- or (S)-stereoisomers or as mixtures thereof. Unless indicated otherwise, the description or naming of a particular compound in the specification and claims is intended to include both individual enantiomers and mixtures, racemic or otherwise, thereof.
• the methods for the determination of stereochemistry and the separation of stereoisomers are well-known in the art (see discussion in Ch. 4 of ADVANCED ORGANIC CHEMISTRY, 4 th edition, March, J., John Wiley and Sons, New York City, 1992).
• the compounds of the present invention may exhibit the phenomena of tautomerism and structural isomerism.
• This invention encompasses any tautomeric or structural isomeric form and mixtures thereof which possess the ability to modulate ITK and JAK3 activity and is not limited to, any one tautomeric or structural isomeric form.
• a compound of the present invention would be metabolized by enzymes in the body of the organism such as human being to generate a metabolite that can modulate the activity of the protein kinases. Such metabolites are within the scope of the present invention.
• a compound of the present invention or a pharmaceutically acceptable salt thereof can be administered as such to a human patient or can be administered in pharmaceutical compositions in which the foregoing materials are mixed with suitable carriers or excipient(s).
• suitable carriers or excipient(s) include REMINGTON'S PHARMACOLOGICAL SCIENCES, Mack Publishing Co., Easton, PA, latest edition.
• a "pharmaceutical composition” refers to a mixture of one or more of the compounds described herein or pharmaceutically acceptable salts or prodrugs thereof, with other chemical components, such as pharmaceutically acceptable excipients.
• the purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
• “Pharmaceutically acceptable excipient” refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
• “Pharmaceutically acceptable salt” refers to those salts which retain the biological effectiveness and properties of the parent compound.
• Such salts may include: (1) acid addition salt which is obtained by reaction of the free base of the parent compound with inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, sulfuric acid, and perchloric acid and the like, or with organic acids such as acetic acid, oxalic acid, (D)- or (L)-malic acid, maleic acid, methanesulfonic acid, ethanesulfonic acid, p- toluenesulfonic acid, salicylic acid, tartaric acid, citric acid, succinic acid or malonic acid and the like, preferably hydrochloric acid or (L)-malic acid; or (2) salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth
• the compound of the present invention may also act, or be designed to act, as a prodrug.
• a "prodrug” refers to an agent, which is converted into the parent drug in vivo. Prodrugs are often useful because, in some situations, they may be easier to administer than the parent drug. They may, for instance, be bioavailable by oral administration whereas the parent drug is not. The prodrug may also have improved solubility in pharmaceutical compositions over the parent drug.
• An example, without limitation, of a prodrug would be a compound of the present invention, which is, administered as an ester (the "prodrug"), phosphate, amide, carbamate, or urea.
• “Therapeutically effective amount” refers to that amount of the compound being administered which will relieve to some extent one or more of the symptoms of the disorder being treated.
• a therapeutically effective amount refers to that amount which has the effect of: (1) reducing the size of the tumor; (2) inhibiting tumor metastasis; (3) inhibiting tumor growth; and/or (4) relieving one or more symptoms associated with the cancer.
• a therapeutically effective amount refers to that amount which has the effect of reducing the pain, temperature, and/or swelling symptomatic of inflammation, both locally or generally.
• disease means any disease or other deleterious condition in which an ITK or JAK3 is known to play a role.
• the term “disease” also means those diseases or conditions that are alleviated by treatment with ITK or JAK3 modulators. Such conditions include, without limitation, cancer and other hyperproliferative disorders as well as inflammation.
• the cancer is a cancer of colon, breast, stomach, prostate, pancreas, or ovarian tissue.
• Such disease includes those associated with abnormal cell growth such as autoimmune, inflammation, rheumatoid arthritis, systemic lupus erythematosus, atherosclerosis, ulcerative colitis, psoriatic arthritis, psoriasis, and Crohn's.
• ITK or JAK3 activity-mediated condition or “disease”, as used herein, means any disease or other deleterious condition in which ITK or JAK3 activity is known to play a role.
• ITK or JAK3 activity-mediated condition also means those diseases or conditions that are alleviated by treatment with an ITK or JAK3 inhibitor.
• administer refers to the delivery of an inventive compound or of a pharmaceutically acceptable salt thereof or of a pharmaceutical composition containing an inventive compound or a pharmaceutically acceptable salt thereof of this invention to an organism for the purpose of prevention or treatment of a protein kinase-related disorder.
• Suitable routes of administration may include, without limitation, oral, rectal, transmucosal or intestinal administration or intramuscular, subcutaneous, intramedullary, intrathecal, direct intraventricular, intravenous, intravitreal, intraperitoneal, intranasal, or intraocular injections.
• the preferred routes of administration are oral and intravenous.
• one may administer the compound in a local rather than systemic manner for example, via injection of the compound directly into a solid tumor, often in a depot or sustained release formulation.
• one may administer the drug in a targeted drug delivery system, for example, in a liposome coated with tumor-specific antibody. In this way, the liposomes may be targeted to and taken up selectively by the tumor.
• compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
• compositions for use in accordance with the present invention may be formulated in any conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
• the compounds of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer.
• physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer.
• penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
• the compounds can be formulated by combining the active compounds with pharmaceutically acceptable carriers well known in the art.
• Such carriers enable the compounds of the invention to be formulated as tablets, pills, lozenges, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient.
• Pharmaceutical preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding other suitable auxiliaries if desired, to obtain tablets or dragee cores.
• Useful excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol, cellulose preparations such as, for example, maize starch, wheat starch, rice starch and potato starch and other materials such as gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl- cellulose, sodium carboxymethylcellulose, and/or polyvinyl-pyrrolidone (PVP).
• disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid. A salt such as sodium alginate may also be used.
• Dragee cores are provided with suitable coatings.
• suitable coatings For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
• Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
• Pharmaceutical compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
• the push-fit capsules can contain the active ingredients in admixture with filler such as lactose, a binder such as starch, and/or a lubricant such as talc or magnesium stearate and, optionally, stabilizers.
• the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Stabilizers may be added in these formulations, also.
• suitable liquids such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
• Stabilizers may be added in these formulations, also.
• Pharmaceutical compositions which may also be used include hard gelatin capsules.
• the capsules or pills may be packaged into brown glass or plastic bottles to protect the active compound from light.
• the containers containing the active compound capsule formulation are preferably stored at controlled room temperature (15-30°C).
• the compounds for use according to the present invention may be conveniently delivered in the form of an aerosol spray using a pressurized pack or a nebulizer and a suitable propellant, e.g., without limitation,
• the dosage unit may be controlled by providing a valve to deliver a metered amount.
• Capsules and cartridges of, for example, gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
• the compounds may also be formulated for parenteral administration, e.g., by bolus injection or continuous infusion.
• Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
• the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulating materials such as suspending, stabilizing and/or dispersing agents.
• compositions for parenteral administration include aqueous solutions of a water soluble form, such as, without limitation, a salt, of the active compound.
• suspensions of the active compounds may be prepared in a lipophilic vehicle.
• Suitable lipophilic vehicles include fatty oils such as sesame oil, synthetic fatty acid esters such as ethyl oleate and triglycerides, or materials such as liposomes.
• Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
• the suspension may also contain suitable stabilizers and/or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
• the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.
• a suitable vehicle e.g., sterile, pyrogen-free water
• the compounds may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
• the compounds may also be formulated as depot preparations. Such long acting formulations may be administered by implantation (for example, subcutaneous ly or intramuscularly) or by intramuscular injection.
• a compound of this invention may be formulated for this route of administration with suitable polymeric or hydrophobic materials (for instance, in an emulsion with a
• a non-limiting example of a pharmaceutical carrier for the hydrophobic compounds of the invention is a cosolvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer and an aqueous phase such as the VPD cosolvent system.
• VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol.
• the VPD cosolvent system (VPD: D5W) consists of VPD diluted 1 : 1 with a 5% dextrose in water solution.
• This cosolvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration.
• the proportions of such a cosolvent system may be varied considerably without destroying its solubility and toxicity characteristics.
• identity of the cosolvent components may be varied: for example, other low-toxicity nonpolar surfactants may be used instead of polysorbate 80, the fraction size of polyethylene glycol may be varied, other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone, and other sugars or polysaccharides may substitute for dextrose.
• hydrophobic pharmaceutical compounds may be employed.
• Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs.
• certain organic solvents such as
• dimethylsulfoxide also may be employed, although often at the cost of greater toxicity.
• the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent.
• sustained-release materials have been established and are well known by those skilled in the art.
• Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days.
• additional strategies for protein stabilization may be employed.
• compositions herein also may comprise suitable solid or gel phase carriers or excipients.
• suitable solid or gel phase carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
• ITK and JAK3 -modulating compounds of the invention may be provided as physiologically acceptable salts wherein the claimed compound may form the negatively or the positively charged species.
• salts in which the compound forms the positively charged moiety include, without limitation, quaternary ammonium (defined elsewhere herein), salts such as the hydrochloride, sulfate, carbonate, lactate, tartrate, malate, maleate, succinate wherein the nitrogen atom of the quaternary ammonium group is a nitrogen of the selected compound of this invention which has reacted with the appropriate acid.
• Salts in which a compound of this invention forms the negatively charged species include, without limitation, the sodium, potassium, calcium and magnesium salts formed by the reaction of a carboxylic acid group in the compound with an appropriate base (e.g.
• NaOH sodium hydroxide
• KOH potassium hydroxide
• Ca(OH) 2 calcium hydroxide
• compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an amount sufficient to achieve the intended purpose, e.g., the modulation of protein kinase activity and/or the treatment or prevention of a protein kinase-related disorder.
• a therapeutically effective amount means an amount of compound effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated.
• the therapeutically effective amount or dose can be estimated initially from cell culture assays. Then, the dosage can be formulated for use in animal models so as to achieve a circulating concentration range that includes the IC5 0 as determined in cell culture (i.e., the concentration of the test compound which achieves a half-maximal inhibition of the ITK or JAK3, or surrogate marker activity). Such information can then be used to more accurately determine useful doses in humans.
• Toxicity and therapeutic efficacy of the compounds described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the IC 50 and the LD 50 (both of which are discussed elsewhere herein) for a subject compound.
• the data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
• the dosage may vary depending upon the dosage form employed and the route of administration utilized.
• the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See, e.g., GOODMAN & GILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, Ch. 3, 9 th ed., Ed. by Hardman, J., and Limbard, L., McGraw-Hill, New York City, 1996, p.46.)
• Dosage amount and interval may be adjusted individually to provide plasma levels of the active species which are sufficient to maintain the kinase modulating effects. These plasma levels are referred to as minimal effective concentrations (MECs).
• MEC minimal effective concentrations
• the MEC will vary for each compound but can be estimated from in vitro data, e.g., the concentration necessary to achieve 50-90% inhibition of ITK or JAK3, or surrogate marker may be ascertained using the assays described herein. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. HPLC assays or bioassays can be used to determine plasma concentrations.
• Dosage intervals can also be determined using MEC value.
• Compounds should be administered using a regimen that maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90%.
• the therapeutically effective amounts of compounds of the present invention may range from approximately 2.5 mg/m 2 to 1500 mg/m 2 per day. Additional illustrative amounts range from 0.2-1000 mg/qid, 2-500 mg/qid, and 20-250 mg/qid. [00107] In cases of local administration or selective uptake, the effective local area
• concentration of the drug may not be related to plasma concentration, and other procedures known in the art may be employed to determine the correct dosage amount and interval.
• compositions administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
• compositions may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient.
• the pack may for example comprise metal or plastic foil, such as a blister pack.
• the pack or dispenser device may be accompanied by instructions for administration.
• the pack or dispenser may also be accompanied by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or of human or veterinary administration.
• Such notice for example, may be of the labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.
• compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
• Suitable conditions indicated on the label may include treatment of a tumor, inhibition of angiogenesis, treatment of fibrosis, diabetes, and the like.
• the compounds and compositions of the invention will find utility in a broad range of diseases and conditions mediated by protein kinases, including diseases and conditions mediated by ITK or JAK3 activity.
• diseases may include by way of example and not limitation, cancers such as lung cancer, NSCLC (non small cell lung cancer), oat-cell cancer, bone cancer, pancreatic cancer, skin cancer, dermatofibrosarcoma protuberans, cancer of the head and neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, colo-rectal cancer, cancer of the anal region, stomach cancer, colon cancer, breast cancer, gynecologic tumors (e.g., uterine sarcomas, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina or carcinoma of the vulva), Hodgkin's Disease, hepatocellular cancer, cancer of the esophagus, cancer of the
• the inventive compound can be used in combination with one or more other chemotherapeutic agents.
• the dosage of the inventive compounds may be adjusted for any drug-drug reaction.
• the chemotherapeutic agent is selected from the group consisting of mitotic inhibitors, alkylating agents, anti-metabolites, cell cycle inhibitors, enzymes, topoisomerase inhibitors such as CAMPTOSAR (irinotecan), biological response modifiers, anti-hormones, antiangiogenic agents such as MMP-2, MMP-9 and COX-2 inhibitors, anti-androgens, platinum coordination complexes (cisplatin, etc.), substituted ureas such as hydroxyurea; methylhydrazine derivatives, e.g., procarbazine; adrenocortical suppressants, e.g., mitotane, aminoglutethimide, hormone and hormone antagonists such as the adrenocorticosteriods (e.g., prednisone), pro
• alkylating agents that the above method can be carried out in combination with include, without limitation, fluorouracil (5-FU) alone or in further combination with leukovorin; other pyrimidine analogs such as UFT, capecitabine, gemcitabine and cytarabine, the alkyl sulfonates, e.g., busulfan (used in the treatment of chronic granulocytic leukemia), improsulfan and piposulfan; aziridines, e.g., benzodepa, carboquone, meturedepa and uredepa; ethyleneimines and methylmelamines, e.g., altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolmelamine; and the nitrogen mustards, e.g., chlorambucil (used in the treatment of chronic lymphocytic leukemia, primary macroglobulinemia and non-Hodgkin's lympho
• antimetabolite chemotherapeutic agents that the above method can be carried out in combination with include, without limitation, folic acid analogs, e.g., methotrexate (used in the treatment of acute lymphocytic leukemia, choriocarcinoma, mycosis fungiodes, breast cancer, head and neck cancer and osteogenic sarcoma) and pteropterin; and the purine analogs such as mercaptopurine and thioguanine which find use in the treatment of acute granulocytic, acute lymphocytic and chronic granulocytic leukemias.
• methotrexate used in the treatment of acute lymphocytic leukemia, choriocarcinoma, mycosis fungiodes, breast cancer, head and neck cancer and osteogenic sarcoma
• pteropterin pteropterin
• purine analogs such as mercaptopurine and thioguanine which find use in the treatment of acute granuloc
• Examples of natural product-based chemotherapeutic agents that the above method can be carried out in combination with include, without limitation, the vinca alkaloids, e.g., vinblastine (used in the treatment of breast and testicular cancer), vincristine and vindesine; the epipodophyllotoxins, e.g., etoposide and teniposide, both of which are useful in the treatment of testicular cancer and Kaposi's sarcoma; the antibiotic chemotherapeutic agents, e.g., daunorubicin, doxorubicin, epirubicin, mitomycin (used to treat stomach, cervix, colon, breast, bladder and pancreatic cancer), dactinomycin, temozolomide, plicamycin, bleomycin (used in the treatment of skin, esophagus and genitourinary tract cancer); and the enzymatic chemotherapeutic agents such as L-asparaginase.
• the vinca alkaloids
• An inventive compound can also be used with other signal transduction inhibitors, such as agents that can inhibit EGFR (epidermal growth factor receptor) responses, such as EGFR antibodies, EGF antibodies, and molecules that are EGFR inhibitors; VEGF (vascular endothelial growth factor) inhibitors; and erbB2 receptor inhibitors, such as organic molecules or antibodies that bind to the erbB2 receptor, such as HERCEPTIN (Genentech, Inc., South San Francisco, CA).
• EGFR inhibitors are described in, for example in WO 95/19970, WO 98/14451, WO 98/02434, and U.S. Pat. No. 5,747,498 and such substances can be used in the present invention as described herein.
• EGFR-inhibiting agents include, but are not limited to, the monoclonal antibodies C225 and anti-EGFR 22Mab (ImClone Systems, Inc., New York, NY), the compounds erlotinib (OSI Pharmaceuticals, Inc., Melville, NY), ZD-1839 (AstraZeneca), BIBX-1382 (Boehringer Ingelheim), MDX-447 (Medarex Inc., Annandale, NJ), and OLX-103 (Merck & Co., Whitehouse Station, NJ), and EGF fusion toxin (Seragen Inc., Hopkinton, MA).
• VEGF inhibitors for example SU-5416 and SU-6668 (Sugen Inc., South San Francisco, CA), can also be combined with an inventive compound.
• VEGF inhibitors are described in, for example, WO 01/60814 A3, WO 99/24440, PCT International Application PCT/IB99/00797, WO 95/21613, WO 99/61422, U.S. Pat. No. 5,834,504, WO 01/60814, WO 98/50356, U.S. Pat. No. 5,883, 113, U.S. Pat. No. 5,886,020, U.S. Pat. No.
• VEGF inhibitors useful in the present invention are IM862 (Cytran Inc., Kirkland, WA); anti-VEGF monoclonal antibody of Genentech, Inc.; and angiozyme, a synthetic ribozyme from Ribozyme (Boulder, CO) and Chiron (Emeryville, CA). These and other VEGF inhibitors can be used in the present invention as described herein.
• pErbB2 receptor inhibitors such as GW-282974 (Glaxo Wellcome pic), and the monoclonal antibodies AR-209 (Aronex Pharmaceuticals Inc., The Woodlands, TX) and 2B-1 (Chiron), can furthermore be combined with an inventive compound, for example, those indicated in WO 98/02434, WO 99/35146, WO 99/35132, WO 98/02437, WO 97/13760, WO 95/19970, U.S. Pat. No. 5,587,458 and U.S. Pat. No. 5,877,305, which are all hereby incorporated herein in their entireties by reference. ErbB2 receptor inhibitors useful in the present invention are also described in U.S.
• An inventive compound can also be used with other agents useful in treating cancer, including, but not limited to, agents capable of enhancing antitumor immune responses, such as CTLA4 (cytotoxic lymphocyte antigen 4) antibodies, and other agents capable of blocking CTLA4; and anti-proliferative agents such as other farnesyl protein transferase inhibitors, for example the farnesyl protein transferase inhibitors described in the references cited in the "Background" section, of U.S. Pat. No., 6,258,824 Bl .
• the above method can also be carried out in combination with radiation therapy, wherein the amount of an inventive compound in combination with the radiation therapy is effective in treating the above diseases. Techniques for administering radiation therapy are known in the art, and these techniques can be used in the combination therapy described herein. The administration of the compound of the invention in this combination therapy can be determined as described herein.
• the Examples depicted below are compounds prepared according to general procedures given in the following sections. Although the synthetic methods and Schemes depict the syntheses of certain compounds of the present invention, the methods and other methods known to one of ordinary skill in the art can be applied to all the compounds of the genus, the genus sub-class and species of each of these compounds as described herein. All aspects of this invention can be understood from the following
• TLC analyses were performed on silica F254 and detection by UV light at 254nm, or by spraying with phosphomolybdic-H 2 S04 dyeing reagent, KMNO4 or iodine.
• Column chromatography were performed on silica Gel 60 (230 mesh). Purifications and separations were performed on a standard silica flash chromatography system. The purity of the samples has been determined by HPLC for the % area peak corresponding to the retention of compound and elemental analysis for C, H, N and O was carried out using Perkin-Elmer 2400 elemental analyser and chloride analysis performed using calorimetric titration at the Intertek USA Inc., QTI.
• toluene/Ethanol (4: lmL) was added a 2 CC>3 (111.69mg, 1.02mmol) the reaction was degassed and purged with nitrogen for lOmin.
• Pd(dppf)Ci 2 (20.7mg, 0.025mmol) was added to the reaction, which was degassed and purged with nitrogen for another lOmin.
• the reaction was heated to 90°C under sealed condition overnight.
• the reaction mixture was allowed to cool to RT and diluted with chloroform.
• the organic layer was filtered through Celite and concentrated to get the crude, which was purified through flash chromatography by using 100-200 mesh silica gel.
• the compound 16 was eluted at 1% methanol in chloroform as off-white solid.
• EXAMPLE_8 (E)-N-(3-(3-methyl-lH-pyrrolo[2,3-b]pyridin-5-yl)phenyl)pent-2-enamide (26):
• toluene/ethanol (4: 1) was added sodium carbonate (111.69mg, 1.02mmol).
• the reaction was degassed and purged with nitrogen for lOmin and Pd(dppf)Ci2 (20.8mg, 0.0255mmol) added to the reaction.
• the reaction was again degassed and purged with nitrogen for lOmin.
• the reaction was heated to 80°C overnight under sealed condition.
• the reaction mass was allowed to cool to rt and diluted with chloroform.
• the organic layer was passed through Celite bed and organic layer was concentrated to get the crude, which was purified through flash chromatography by using 100-200 mesh silica gel.
• the compound 41 was eluted at 30% ethyl acetate in hexane as off white colour solid.
• toluene/ethanol (4: 1) was added sodium carbonate (111.69mg, 1.02mmol).
• the reaction was degassed and purged with nitrogen for lOmin.
• Pd(dppf)Ci2 (20.8mg, 0.0255mmol) was added to the reaction.
• the reaction was again degassed and purged with nitrogen for lOmin.
• the reaction was heated to 80°C overnight under sealed condition.
• the reaction mass was allowed to cool to rt and diluted with chloroform.
• the organic layer was passed through Celite bed and organic layer was concentrated to get the crude, which was purified through flash chromatography by using 100-200 mesh silica gel.
• the compound 44 was eluted at 30% ethyl acetate in hexane as off white colour solid.
• the reaction was heated to 90°C under sealed condition overnight, allowed to cool to rt, and diluted with chloroform .
• the organic layer was filtered through Celite bed, concentrated to get the crude, which was purified through flash chromatography by using 100-200 mesh silica gel.
• the compound was eluted in 3% methanol in dichloromethane as pale yellow colour solid 97.
• the reaction was degassed and purged with nitrogen for another lOmin, heated to 90°c under sealed condition overnight, allowed to cool to rt, and diluted with chloroform .
• the organic layer was filtered through Celite plug and concentrated to get the crude, which was purified through flash chromatography by using 100-200 mesh silica gel. The compound was eluted in 5% ethyl acetate in hexane as off-white solid 99.
• the reaction was heated to 90°C under sealed condition overnight, allowed to cool to rt, and diluted with chloroform .
• the organic layer was filtered through Celite bed, concentrated to get the crude, which was purified through flash chromatography by using 100-200 mesh silica gel.
• the compound was eluted in 3% methanol in dichloromethane as pale yellow colour solid 101.
• the reaction mixture was allowed to cool to room temperature, diluted with chloroform.
• the organic layer was filtered through celite plug and concentrated to get the crude compound 135.
• the crude was purified through flash chromatography by using 100-200 mesh silica gel.
• the compound was eluted in 5% ethyl acetate in hexane as half white solid 5-bromo-3-(4-fluoro-2-methoxy- 5-methylphenyl)-l-tosyl-lH-pyrrolo[2,3-b]pyridine 135.
• the reaction mixture was allowed to cool to room temperature, diluted with chloroform.
• the organic layer was filtered through celite plug and concentrated to get the crude compound 136.
• the crude was purified through flash chromatography by using 100-200 mesh silica gel.
• the compound was eluted in 5% ethyl acetate in hexane as half white solid 3-(3-(4-fluoro-2-methoxy-5- methylphenyl)-l-tosyl-lH-pyrrolo[2,3-b]pyridin-5-yl)aniline 136.
• the reaction mixture was allowed to cool to room temperature, diluted with chloroform .
• the organic layer was filtered through celite plug and concentrated to get the crude .
• the crude was purified through flash chromatography by using 100-200 mesh silica gel. The compound was eluted in 5% ethyl acetate in hexane as half white solid 143.
• a solution of 143 (200 mg, 0.424 mmol) was dissolved in THF/DMF was added diethyl amine (67.8 mg, 0.848 mmol).
• EDC.HC1 (131.44 mg, 0.848 mmol) and 131 (139.9 mg, 0.848 mmol) was added to the reaction and stirred at RT for overnight. After completion, the reaction was diluted with water and the aqueous was extracted with 10% methanol in chloroform for two times. The organic layer was dried over sodium sulphate and concentrated to get the crude. The crude was purified through neutral alumina, and the compound was eluted at 2% methanol in chloroform as half white colour compound 144.
• a solution of 144 (100 mg, 0.171 mmol) was taken in methanol (7 ml) and water (3 mL) was added potassium carbonate (47.4 mg, 0.343 mmol). The reaction was heated to 60 °C for overnight. The methanol was completely distilled and diluted with water. The organic phase was extracted with ethyl acetate (50 mL) twice. The organic layer was dried over sodium sulphate and filtered and concentrated to get the crude. The crude was purified through flash chromatography by using neutral alumina.
• the reaction mixture was allowed to cool to room temperature, diluted with chloroform .
• the organic layer was filtered through celite plug and concentrated to get the crude .
• the crude was purified through flash chromatography by using 100-200 mesh silica gel. The compound was eluted in 5% ethyl acetate in hexane as half white solid 143.
• the organic layer was filtered through celite plug and concentrated to get the crude .
• the crude was purified through flash chromatography by using 100-200 mesh silica gel. The compound was eluted in 5% ethyl acetate in hexane as half white solid 143.
• Pd(dppf)Ci2 (16.88 mg, 0.0206 mmol) was added to the reaction. The reaction was degassed and purged with nitrogen for another 10 min. The reaction was heated to 90 °C under sealed condition for overnight. The reaction mixture was allowed to cool to room temperature, diluted with chloroform. The organic layer was filtered through celite plug and concentrated to get the crude. The crude was purified through flash chromatography by using 100-200 mesh silica gel. The compound was eluted in ethyl acetate in hexane as half white solid 3-(3- methyl-lH-pyrrolo[2,3-b]pyridin-5-yl)aniline 24.
• a solution of 106 (200 mg) was taken in methanol (7 mL) and water (3 mL) was added potassium carbonate (100 mg). The reaction was heated to 60 °C for overnight. The methanol was completely distilled and diluted with water. The organic phase was extracted with ethyl acetate (50 ml) twice. The organic layer was dried over sodium sulphate and filtered and concentrated to get the crude. The crude was triturated with hexane to afford the half white colour solid 107.
• the organic layer was filtered through celite plug and concentrated to get the crude .
• the crude was purified through flash chromatography by using 100-200 mesh silica gel. The compound was eluted in 12% ethyl acetate in hexane as half white solid 166.
• a solution of 167 (50 mg, 0.130 mmol) was dissolved in dichloromethane (10 mL) and was cooled to 0 °C. Triethyl amine (19.7 mg, 0.195 mmol) was added to the reaction mass and kept for stirring. Acrolyl chloride 146 (14mg, 0.156 mmol) was added drop wise to the reaction mass and kept stirring for 4 hr. After completion reaction was quenched with water and the organic layer was separated and aqueous phase was again extracted with DCM. The combined organic layer was washed with brine solution. The organic layer was dried over sodium sulphate and concentrated to get the crude.
• ITK and JAK3 Kinase assay procedures Enzyme was incubated with substrate peptide in reaction buffer in the presence and absence of test compounds or Staurosporine. All additions were done on ice, followed by the addition of ATP mix. Wells were uniformly mixed using an Eppendorff plate shaker and incubated at 30°C for 20min, and stopped by the addition of 5 ⁇ ⁇ of 3% phosphoric acid. Volume was increased to ⁇ , by adding 0.8% phosphoric acid which was then transferred to PC filter mats (Millipore), pre-equilibrated with 70% ethanol and water. Plates were washed thrice with ⁇ , 0.8% phosphoric acid and dried for an hour at 60°C.
• ⁇ scintillation fluid was added into each well and reading taken in Perkin Elmer TOPCOU T beta counter. The data analysis was performed by averaging the duplicate top count readings for each standard, negative, positive control (enzyme control) and samples and subtracting the average negative control from each reading which results in corrected values.
• a validation EC5 0 curve was generated by plotting CPM for each Staurosporine concentration on y-axis against the Log concentration of Staurosporine (nM) on the x-axis followed by a best fit curve through the points.
• Table 3B List of Compounds and Corresponding ITK and JAK3 kinases tested : 140 * **
• Dose formulations were prepared on the day of doing. Blood samples were collected at 0.083 (only IV), 0.25, 0.5, 1, 2, 4, 8 and 24 h post-dose. At each time point, approximately 0.2mL of blood was withdrawn from each cannulated rat through jugular vein and transferred to a pre-labeled microfuge tube containing 20 ⁇ 1 ⁇ of 200mm K 2 EDTA permL of blood. Following collection of blood sample, equal volume of heparinized saline was flushed into jugular vein of rat. The blood samples were centrifuged at 5000g for 5min at 4 ⁇ 2°C. The plasma was separated within 30min of scheduled time and stored below -60°C until bio-analysis.
• the plasma samples were analyzed for selected test EXAMPLES using a fixxxt- for purpose liquid chromatographic tandem mass spectrometric detection (LC-MS/MS) method with a lower limit of quantification of 2.21 ng/mL.
• the pharmacokinetic parameters for select EXAMPLES were calculated using the non-compartmental analysis tool of validated WinNonlin ® software (Version 5.2).
• lH-pyrrolo[2,3-b]pyridine, lH-pyrazolo[3,4-b]pyridine and 5H- pyrrolo[2,3- b]pyrazine series of compounds its analogs series were designed using X-ray crystal structural models of JAK3 and ITK. Based on the 3-D profile scoring, the structural template was chosen from PDB database JAK3 (3ZEP) and ITK (3MJ2). Several models were built and refined to check the 3D profile and implemented the FIELDS technology lead to the successful discovery and claim oflH-pyrrolo[2,3-b]pyridine series of compounds.
• Table 7 lH-pyrrolo[2,3-£]pyridine class of Janus and TEC Kinase Selectivity.
• PLC- ⁇ Inhibition - Calcium Efflux (FLIPER) Assay To determine the pharmacodynamics effect of covalent ITK inhibition, T cells were stimulated at various times following ITK inhibition and the phosphorylation of PLCyl was measured. lH-pyrrolo[2,3- &]pyridine series of compounds found to be inhibitors of PLCyl mediated calcium release from CD4+ T cells via TCR engagement (cellular ITK inhibition). This was corroborated by the IC S e s with lH-pyrrolo[2,3-/?]pyridine series of compounds and found to be 630 nM
• ITK The T Cell Receptor (TCR) pathway is significantly effected when NFAT cellular profiling assay was performed where lH-pyrrolo[2,3-/?]pyridine series is inhibited with an IC 50 of 176 nM and Tofacitinib (CP-690550) had > 10 uM. This data is a direct read out for ITK inhibition mechanism for lH-pyrrolo[2,3-/?]pyridine series. Additionally PLCy phosphorylation data further supports the ITK pharmacology.
• TCR T Cell Receptor
• lH-pyrrolo[2,3-/?]pyridine series of compounds Mice IL-2, IL-4 and IFN-g: 60 mg/Kg is highly efficacious. There is a significant increase in serum IL-2, IL-4 and IFN- gamma (P ⁇ 0.001) of Positive Control animals compared to negative control animals. 1H- pyrrolo[2,3-/?]pyridine series of compounds are not significantly decreased serum IL-2, IL-4 and IFN-gamma at doses used when compared to Positive control. However, at 60 mg/Kg dose lH-pyrrolo[2,3-Z?]pyridine series of compounds significantly reduced IFN-g production.
• Reference compound CP-690550 has shown significant decrease in serum IL-2, IL-4 and IFN-gamma at 1.5 hrs post-antibody treatment.
• Dexamethasone has shown significant decrease in serum IL-2, IL-4 and IFN-gamma at 1.5 hrs post-antibody treatment.
• lH-pyrrolo[2,3-/?]pyridine series of compounds was dosed in BID study due to its 4 times higher solubility over non-salt form.
• lH-pyrrolo[2,3-Z?]pyridine series of compounds at dose 100 mg/Kg is well tolerated with no clinical signs.
• Tofacitinib at 60 mg/Kg had body weight change from day 1 and this is the highest dose recommended for Tofacitinib and cannot be dosed over 60 mg due to solubility issues.
• the body weight is generally a direct reflection of efficacy, the more efficacious the treatment the less body weight (bw) loss. This does not hold true in instances of overt toxicity.
• the bw increase in the 100 mg/kg group of lH-pyrrolo[2,3-Z?]pyridine series of compounds is a reflection of increased efficacy (animals are able to move better, and have a more normal appetite, more normal water consumption during the schedule), lH-pyrrolo[2,3-Z?]pyridine series of compounds efficacy by attenuating paw arthritis scores (swelling, edema and paw volumes).
• the CIS study is an established/chronic CIA model study we conducted where 1H- pyrrolo[2,3-Z?]pyridine series of compounds highly efficacious similar or higher over Tofacitinib and no infections were seen.

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